- Email: [email protected]
RNA and protein synthesis required for entry of cells into mitosis and during the mitotic cycle
Q 1065 1)~ ~\cademic Esperimenlal
Press Inc.
Cell Research
46,
93-105
93
(1967)
RNA AND PROTEIN SYNTHESIS REQUIRED FOR ENTRY OF CELLS INTO MITOSIS AND DURING THE MITOTIC CYCLE GRACE Department
of Biology,
M.
DONNELLY City
of Hope
Received
and Medical August
J.
E.
Center,
SISKEN Duarte,
CaliJ,
U.S.A.
29,1966
THE fact that there is a delay between the completion of DNA synthesis and the entry of cells into recognizable mitosis in nearly all somatic cells thus far studied suggests, a priori, that this is a period during which specific metabolic or organizational events related to the ensuing division are occurring. It also suggests that there is some relationship between the completion of DNA synthesis and the initiation of those events which convert an interphase cell into one organized for division. Inhibitors whose primary modes of action in cells are relatively well defined, when used in conjunction with various kinetic techniques, afford a means of studying biochemical events at different times during the mitotic cycle. Thus, by using puromycin, an inhibitor of protein synthesis [as], and actinomycin D (AMD), an inhibitor of DNA-dependent RNA synthesis [S], it is possible to study the requirements for division of the synthesis of nucleic acids and proteins during the predivision period. Indeed, numerous such studies have recently been reported [2, 10, 17, 18, 24-271. However, thus far, none has exploited Perry’s [19] finding that treatment with AMD can lead to qualitatively different effects depending on the levels of the agent utilized; at 4 x 1O-8 M the main inhibitory effect of actinomycin D is on ribosomal RNA synthesized at the nucleolus, while at 4 x 10-s M all RNA synthesis is inhibited. In this study we attempted to gain some insight into the roles of the RNAs synthesized during the predivision period by comparing the effects of these concentrations of actinomycin D to the effects of puromycin and by studying the characteristics of cell populations which had been exposed to actinomycin D for short periods and then returned to normal medium. Contribution No. G2-66, Department of Biology. Supported from the National Cancer Institute, U.S. Public Health Service Grant FR 05471, U.S. Public Health Service.
in part by Grant CA-04526-07 and General Research Support
Experimental
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94
Grace M. Donnelly
and J. E. Sisken
METHODS The Fernandes line of human amnion cells was used in all of our experiments. These cells were grown in Rose chambers or on coverslips in Leighton tubes. All coverslips were fixed with two changes of ethanol acetic acid, 3:1, and rinsed with two changes of ethanol. For mitotic index studies, the cells were stained with acetic orcein, dehydrated in ethanol, and mounted with Permount. Coverslips for autoradiographs and liquid scintillation counting were further extracted with several changes of cold 5 per cent TCA followed by several rinses in distilled water. Kodak NTB-2 emulsion was used for autoradiographs which were stained through the film with Azure A after development. Actinomycin D (generously supplied by Dr. George E. Boxer, Merck and Co., Rahway, New Jersey) was used in two concentrations, 4 pg/ml (approximately 4 x 1O-s M) and 0.04 pg/ml (approximately 4 x 1O-8 M). Puromycin was used at a concentration of 25 lug/ml (4.6 x 1O-6 M). Tritiated uridine was used at a concentration of 2 PC/ml (3c/mM). DNAse (Worthington, 1 x crystallized, 0.1 mg/ml, pH 7.5) digestions were performed at room temperature for 3 hr [ll]. RNAse (Worthington, crystallized from EtOH, 2 mg/ml, pH 6) digestions were performed at 45°C for 3 hr [15]. The cinemicrographic method was as previously described [al], using low-power phase objectives and with exposures being made at a rate of one frame per minute. For measurements of gross rates of synthesis, coverslips with labelled cells were crushed in glass counting vials. The cells were solubilized by shaking with Hyamine 10X for l& hr at 37°C. Counting fluid was added and the radioactivity was measured in a liquid scintillation counter. RESULTS The effects of the inhibition
of protein
synthesis
The effects of 4.6x 1W6 M puromycin on three cell parameters are shown in Fig. 1. Curve A shows that this concentration reduces the rate of protein synthesis in our material to about a third of the control rate within 15-30 min, levels off quickly and remains at the 20-25 per cent level for at least 2 hr. No samples were taken beyond this time, since time-lapse motion pictures of cells in Rose chambers showed that cells began to die about 2 hr after the beginning of treatment. The time-lapse films also showed that cells were inhibited from entering mitosis very soon after exposure to this concentration of puromycin. The kinetics of this inhibition are shown by the data of curve B, Fig. 1, which demonstrates that the metaphase index drops very rapidly to about half the control level in 30 min and to essentially zero in 2 hr. Because of the rapidity of this effect and because cells begin to die within 2 hr, the question arises as to whether the agent caused an overall inhibition of cellular metabolic processes, perhaps through an inhibition of some Experimental
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RNA and protein
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synthesis and mitosis
sensitive step in a generally required system (e.g., some rapidly-turning-over respiratory enzyme), or whether the effect has some degree of specificity on the processes required for the entry of cells into division.We therefore studied the effects of puromycin on the gross rate of nucleic acid synthesis and found (Curve C, Fig. 1) that this level of puromycin inhibits the rate of incorporation of 3H-uridine to only a small extent over a 2-hr period. % CONTROL 130
1
c
Fig.
1.
Fig.
2.
Fig. l.-Effects of 4 x 1O-6 M puromycin on human amnion cells. Curve A, Relative rate of incorporation of isotope into replicate coverslip cultures exposed to W-leucine for 15 min prior to fixation at various intervals after the beginning of treatment. Curve B, The mitotic index at various times since exposure of coverslip cultures to puromycin. Each point is based on counts of 9000 cells from at least 2 experiments. The control value is based on counts of 155,000 cells obtained from several different experiments. Curve C, Same as in curve A, except cells were exposed to SH-uridine. In curves A and C, nearly all points are derived from 3 or 4 replicate cultures in each of at least 2 separate experiments. Curve D, A reproduction of curve B, Fig. 2, metaphase index following treatment with 4 pg/ml AMD. Fig. 2.-Actinomycin inhibition of RNA synthesis and mitosis during treatment. The metaphase index was determined for cells grown on coverslips and exposed to 4 fig/ml (Curve B) and 0.04 pg/ml (Curve D) of AMD for varying time periods before fixation. 5000 to 11,000 cells were counted for each time period. The number of metaphases for each 1000 cells was recorded using a double blind scoring method. Controls were averaged and treatments were compared with the overall control mean (17.1kO.64 metaphases per thousand cells) based on 155,000 cells. Other coverslips were similarly exposed to AMD (Curve A, 4 pg/ml; Curve C 0.04 ,og/ml) but then labelled with tritiated uridine (2 PC/ml) in the appropriate concentration of AMD for 15 min prior to fixation. Fixed cells were further extracted with cold 5 per cent TCA and the coverslips were prepared for liquid scintillation counting. Counts/min/sample from tritiated uridine in actinomycin treated samples were compared with counts/min in untreated control samples. Experimental
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Grace M. Donnelly
The effects of the inhibition
and J. l?. Sisken
of RNA synthesis
The effect of the concentrations of AMD used by Perry [19] on the overall rates of incorporation of 3H-uridine into total nucleic acids are shown in Fig. 2, Curves A and C. After only 10 min of treatment with 4 pg/ml AMD, 3H-uridine incorporation had dropped to almost 30 per cent of the control value and was down to 15 per cent of control after 1 hr. With 0.04 pg/ml, the decrease was not nearly so drastic. Samples treated for 10 min had 65 per cent of the control value, and at the end of 3 hr, incorporation was still at 30 per cent of the control level. Control samples which had been RNAase digested following exposure to 3H-uridine in two experiments indicated that 3H-uridine in the DNA fraction contributed 7-10 per cent to the total counts. The differential effects of these concentrations of AMD on nucleolar and non-nucleolar RNA synthesis are indicated by the autoradiographs of Fig 3. Untreated controls showed the expected short term 3H-uridine labelling pattern (Fig. 3A, B, C). Nuclei were well labelled and there was a large population of cells with heavily labelled nucleoli. Grains in the cytoplasm were not above background, and RNAase digestion resulted in autoradiographs with essentially no label above background in about two-thirds of the cells (Fig. 3B). The remaining cells retained some label, presumably in DNA. Cells pretreated with 4 ,ug/ml AMD have all autoradiographically detectable RNA synthesis blocked within 15 min (Fig. 3F). A loo-fold dilution, on the other hand, blocks nucleolar RNA synthesis selectively and quickly, while chromatin RNA synthesis is relatively unaffected (Fig. 3D). The effect of actinomycin on the entry of cells into mitosis is shown by the metaphase index data of Fig. 2, Curves B and 1). With 4 pg/ml AMD (Curve B), there was a surprisingly rapid decline in the metaphase index, one which was almost as rapid as that produced by puromycin (Fig. 1, Curve B). In fact, the difference between the two curves at the 50 per cent level is of the order of only 10 min (Fig. 1, Curve D). The lower concentration of actinomycin produced a different pattern of mitotic inhibition (Fig. 2, Curve D). The metaphase index of populations treated with 0.04 ,ug/ml remained at about the control value for almost 3
Fig. 3.-Autoradiographs of human amnion cells exposed to tritiated uridine following actinomycin treatment. Cells were treated with AMD (4 pg/ml or 0.04 pg/ml) for 15 min and then labelled for 15 min with tritiated uridine (2 PC/ml) in the appropriate concentration of AMD prior to fixation. Experimental
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KXA and protein
A, W and B, RKAase ,ug/ml A&ID /lg/ml AMD treated with
C, Untreated controls, showing digested. C, DN\‘Aase digested showing selective loss of label and DNAase digested, showing 4 /cg/ml AMD showing almost
synthesis und miiosis
the typical uridine labelling pattern. A, undigested; showing labelled nucleoli. II, Cells treated with 0.04 associated with nucleoli. I:‘, Cells treated with 0.0-1 presence of non-DIV.4 label in nucleus. I:. Cells total loss of label in both nucleolus and chromatin.
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Grace M. Donnelly
and J. E. Sisken
hr, and then dropped to 75 per cent of control at the fourth hour. After seven hours of treatment, the metaphase index had fallen to about 20 per cent, and by the tenth hour, the slides were unscorable. There is some variability in the data and it is formally possible to also fit the line E to these same data. The explanation for such a line would be that an initial increase in metaphase index, due to prolongation of metaphase, occurred immediately after the beginning of treatment, but that there was also an immediate inhibitory effect on the entry of cells into metaphase. However, direct observations with time-lapse cinemicrography showed that the continuous treatment of cells with this concentration of AMD over a much longer period has no effect on the duration of metaphase. During a 24 hr control period prior to treatment, metaphase times from three separate experiments averaged 16.4kO.4 min (n=145) while the mean duration of metaphase for periods of up to 7.5 hr of continuous treatment was 16.4+ 1 .O min (n=45). Since metaphase times are not increased, we believe line D presents a more correct interpretation of the effects of 0.04 pg/ml AMD on metaphase index. Time-lapse cinemicrographs showed that actinomycin killed Fernandes human amnion cells quickly. With continuous exposure to 4 ,ug/ml AhID, cells in the colonies under observation began to die within 2-4 hr after the beginning of treatment. By lo-15 hr, the colonies were usually totally obliterated. Very few divisions were seen after the beginning of treatment at this concentration. Those cells that did divide did so within the first 5 hr of treatment and exhibited a prolonged metaphase (Fig. 4). Death, as defined by active “boiling” of the cell and its subsequent “explosion” or collapse, occurred during interphase. With continuous treatment at the lower concentration, deaths began approximately 3 hr after introduction of the drug, and were widespread by the 7th hr. Divisions after treatment were more common than in the colonies treated with the higher concentration, and were noted as late as 20 hr after the beginning of treatment. At approximately 22 hr, almost all cells were dead. Recovery of cells after removal of actinomycin The fact that under continuous treatment metaphase times were not increased and the metaphase index was relatively unaffected by 0.04 ,ug/ml AMD for up to 3 hr (Fig. 2, Curve D), shows that cells in the last 3 hr of interphase (i.e., late S or G2) were not delayed in their entry into mitosis. The studies in this section were designed to determine the effects of a 3 hr Experimental
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RNA and protein
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treatment at this lower concentration on other aspects of cellular kinetics. (No further studies were made of the higher concentration because the time-lapse films had shown that removal of actinomycin after 3 hr treatment at 4 ,ug/ml AMD did not result in any recovery, and that cells continued to die, although perhaps at a slightly slower rate.) In the first experiments, populations of cells growing in Rose chambers were followed by time-lapse cinemicrography under control conditions for periods of up to 24 hr in order to record mitotic histories of individual cells. Photography continued during the 3 hr treatment with 0.04 pg/ml AMD as well as during the post-treatment period. Cells were followed for periods of up to 95 hr beyond treatment. The data in Fig. 5 indicate the duration of mitotic cycles of cells born at different times in relation to the period of treatment. Cells 17-20 hr old at the time of treatment, and thus in late S or G2, had a normal cycle time. Progeny of these cells, however, had elongated cycle times. Cells in all other parts of the cycle at the time of treatment had elongated cycle times, as did their progeny. Analysis of the films also revealed that a burst of mitosis occurred 20 hr
Fig. Fig. 4.-Prolongation (arrow) represent class,
4.
7 and
Fig. of metaphase during 5 cells, respectively.
treatment with 4 pg/ml There were no divisions
5.
AMD. Points after treatment beyond the 5th hr treatment
Fig. B.-Effect of a short treatment of actinomycin D on the duration of the mitotic cycle of cells born at different times in relation to the time of treatment. Cells in a randomly dividing population were followed cinemicrographically before, during, and after 3 hr treatment with 0.04 ,ug/ml AMD. The chart shows that the generation times of cells is related to the age of the cell at the time of treatment. The length of the generation time is shown in hours. The number of cells in each age class is given in parentheses. The left end of the bar indicates the time of birth, and the right end the time of division relative to the time of treatment. The standard errors for the length of the generation times is indicated by the line at the right end of the bar. Experimental
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Grace M. Donnelly
100
and J. E. Sisken
after treatment and that there was an accompanying increase in the duration of individual metaphases (Fig. 6, Curve A). .4 subsequent mitotic index study, utilizing coverslip cultures and the techniques as described in Fig. 2, substantiated the existence of the 20.hr post-treatment peak. These data are compared in Fig. 6. Studies of recovery of cells following a 3 hr treatment with the low concentration of AMD revealed that the gross rate of RNA synthesis returned to about 75 per cent of the control level (Fig. 6C) almost immediately after
0
.i b” L0
---L,” 10
20 TIME
jo
-407
IHOURS)
Fig. 6.-Mitotic index, RNA synlhrsis, and duration of metaphase following a short-term treatment of actinomycin D at 0.04 pg/ml. -4, Duration of Metaphase (inner scale, in min); B, mitotic index; C, RNA synthesis.
removal of AMD, but that R?;A synthesis did not return to normal until after the tenth hour. Further increase in rate may simply reflect increase in cell number. This level of recovery is greater than that reported by Taylor [X53, but this is probably a result of the lower concentration of AMD used in our experiment. DISCUSSION
The data presented on the efyects of puromycin on the entry of cells into division are in general agreement with previous studies [lo, 25, 261 which indicate that mammalian cells in culture must synthesize at least some protein to within 30-60 min of metaphase in order to carry out those processes which convert them from interphase to mitotic cells. The fact that nucleic acid synthesis is not greatly affected during the first hour or two of treatment with puromycin suggests that the nucleic acid synthesizing systems as well as the systems supplying energy, precursors, and enzymes for nucleic acid Experimental
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RNA and protein
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synthesis and mitosis
synthesis, are not nearly so sensitive to the agent as is the entry of cells into division. It further suggests that the entry of cells into division is dependent upon the synthesis of new protein molecules just prior to mitosis. Reports in the literature concerning the last synthesis of RNA required for cells to reach mitosis in cultured mammalian cells are more varied and, in some cases at least, are almost certainly a result of differences in concentration of AMD used by the different investigators. These experiments are summarized in Table I, and may be divided into two groups: those in which the concentration of ARID ranged between 0.025 pg/ml and 0.2 ,ug/ml, which WC will call a low concentration; and those in which the concentration utilized was 2 to 5 pg/ml, which we will call a high concentration. Perry [lg] showed that concentrations of the order of 0.04 pg/ml inhibit the synthesis of mainly ribosomal RNA, whereas 4 pg/ml inhibits all RNA synthesis in HeLa cells. Since our autoradiographs tend to confirm these results for amnion cells, it appears that one may interpret all the data on lovv concentration, except Taylor’s [25], as showing the elrects of the inhibition of ribosomal RNA. The data from high concentration experiments indicate the requirement for some other RNA, perhaps messenger RNA. In all experiments but Taylor’s [a;], where lovv concentrations of AMD were used, RNA synthesis, when measured, was incompletely inhibited. The time beyond which this concentration was no longer efTective in inhibiting the entry of cells into mitosis vvas placed at 2 to 4 hr prior to division. In some cases this coincided with the end of the S period. Taylor’s experiments were exceptional in that he obtained complete inhibition of RNA synthesis with as 0.2 ,ug/ml of AMD but he did not observe an efrect on the mitotic index until 3 hr after the beginning of treatment. \Vhen the effects of low concentrations of AMD are compared with those of puromycin, it is always found (Table I) that the latter affects metaphase index much more rapidly, This observation suggests that the low concentration of actinomycin causes the level of some RNA fraction to decline to a point where the metabolic requirements for entry of cells into division can no longer be met. While there might be alternative explanations, this eflect may occur because of the direct role of the RNA in the synthesis of a required protein. If this be true and, if the only significant effect of the low concentration of AMD is on ribosome synthesis, then it is possible to conclude from our data as well as from the data of others that, in the latter part of interphase a cell has approximately a l-3 hr supply of ribosomes which can be used for the synthesis of proteins required for division. This possibility is consistent with the findings of Gaulden and Perry [4] and Schiff [20] Experimental
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Grace M. Donnelly
102
and J. E. S&ken
that nucleolar function immediately prior to division is not requisite for that mitosis. Further support for this point is presented below. The experiments on the effects of continuous treatment with the low concentration of actinomycin D on the mitotic index show that only cells in the last part of interphase (late S-G2) at the time of treatment can proceed to division, and that they do so at normal rates. When, after 3 hr of treatment, we rinsed the cells and reincubated them in fresh medium, all observable TABLE I. Time between
Duration of G2
Cell type
treatment with actinomycin cells into mitosis. Concentration actinomycin ielm
Reference
LOW
KB
3 hr
Rabbit
kidney
Mouse Mouse Human
fibroblast fibroblast amnion
Min
of 4 hr
0.2
[lo]
0.33
PI PI
0.075 0.025-0.05 0.04
This
3.2 hr
P61
2.2 hr
[II This
paper
HIGH
Chinese Chinese Human
hamster hamster amnion
Degree of inhibition of RNA synthesis
Time between treatment and inhibition of entry into mitosis
CONCENTRATION
WI
2.2 hr
of D
D and efect on entry of
Essentially total in 15 min 90 yh over a 4-hr period
Approx. 80 y0 in 1 hr
3 hr Approx.
4 hr
2 hr “Considerable 3-4 hr
time”
CONCENTRATIOS
paper
2.0 5.0 4.0
85 % m 1 hr Approx. 95 % in 1 hr
1.3 hr Approx. 45 min 30-40 min
cells continued on to division and, in many cases their progeny could be followed into subsequent divisions. Cells which were in early interphase during the treatment required more than the normal amount of time to reach mitosis. Cells which were in late interphase entered mitosis at their normally expected times, but the progeny of these cells had prolonged cycle times which were approximately equal to the cycle time of those cells which had been treated in the early part of their cycle. These results are similar to observations which have been made on slime molds [18] and protozoa [17, 271. The fact that treatment in the late part of one mitotic cycle can affect the subsequent division is explainable in at least two ways. The first possibility Experimental
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RNA
and protein
103
synthesis and mitosis
is simply as suggested above, namely that the ribosomes made in G2 are really required by the cell in its next cycle and, if the cell begins its next cycle with a deficiency of ribosomes, its Gl and cycle time will be prolonged. It has been shown by Killander and Zetterberg [S] that variations in Gl (which in our cell line accounts for most of the variations in duration of the mitotic cycle [22, 231) are dependent upon the mass of a cell at the beginning of interphase, and that cell mass and rate of protein synthesis during the mitotic cycle are correlated with RNA content [9, 291. On the other hand, it has been shown that AMD binds very tightly to DNA [6j, and it is not easily removed by simple washing. This, then, suggests the second possibility which is that, even though cells in G2 at the time of treatment can divide, they still may have bound to their DNA some A1fD that continues to inhibit the synthesis of RNA throughout the next cycle. These hypotheses are not mutually exclusive, and, in reality, both hypotheses are probably correct to at least some extent. In all three experiments with the high concentration of AMD (Table I) the effect on the entry of cells into division occurs between 0.5 and 1.3 hr after the beginning of treatment. Only in our experiments and those of Tobcy et al. [26] may the effects of the high concentration of AMD be compared with those of puromycin. In the latter the differences in time between the effects of the two agents is about 45 min while in our experiments it is of the order of 10-15 min. As indicated by the differences in G2 times of these two cell lines, the 30-min discrepancy could easily be a result of inherent differences in the cell lines utilized. In either case, however, we need to be able to account for the gap in time between the effects of the two agents. It is possible to propose at least four explanations for this gap: (1) The cell in G2 must continually make some messenger RNA which has a very short half-life. From our data, for example, it would be estimated to be something like 10-15 min. While messengers with half-lives of about a minute have been reported for bacterial cells [14], the shortest half-life reported for a mammalian messenger is about 1 hr [5]. (2) S ome division-related messenger is synthesized up to but not beyond a specific time prior to division (45 min in Chinese hamster cells, and lo-15 min in human amnion). It need not have an exceptionally short half-life. This is the kind of hypothesis favoured by Tobey et al. [26]. (3) There is another kind of RN,4 involved which has the characteristic of messenger RNA in being inhibited only by high concentrations of AMD, but it is not a messenger in the usual sense. A possible example of this might be the RNA which is usually found associated with the mitotic apparatus (see review by Mazia, [16]). (4) The rapid effect of the high Experimental
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Grace M. Donnelly
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concentration in our system is a result of some side reaction of AMD unrelated to RNA synthesis. Actinomycin at high concentrations has been shown to affect other systems, such as energy metabolism [7, 121 and protein synthesis [3, 13, 251 but, even at very high concentrations, the levels of inhibition observed within the first hour of treatment by these authors do not seem nearly great enough to account for the rapid decrease in mitotic index observed in our experiments. To our knowledge, no critical experiments have been reported which would rule out any of these possibilities.
SUMMARY
As a means of studying metabolic requisites for mitosis, actinomycin D, in two concentrations, and puromycin were compared in their respective inhibitions of RNA and protein synthesis during the predivision period. Treatment of the cells with levels of puromycin which reduce the gross rate of protein synthesis to about 20 per cent of the control level rapidly inhibits the entry of cells into mitosis. In agreement with the findings of others, the kinetics of this effect suggest that new protein molecules must be synthesized within a 30-60 min period prior to metaphase for division to occur. Low concentrations of actinomycin (0.04 pg/ml) which selectively inhibit nucleolar RNA synthesis do not affect the entry of cells into division for periods of up to 3 hr. This suggests that cells in the latter part of the mitotic cycle already contain a l-3 hr supply of ribosomes which can be utilized for the syntheses involved in preparing cells for division. Removal of these low concentrations of AMD from the medium after a 3-hr period allows division to occur, but c,ells treated early in their mitotir cycle require more time to get to division. Cells treated during G2 or late S are not delayed in entering division but their next mitotic cycle is prolonged. A high concentration of AMD (4 pg/ml) affects the entry of cells into division almost as rapidly as puromycin. Several alternative possibilities are presented to account for this finding. REFERENCES 1. ARRIGHI, F. E. and Hsu, T. C., Exptl Cell Res. 39, 305 (1965). 2. CASPERSSON, T., FARBER, S.,FOLEY,G.E. and KILLANDER, D., ExptlCeZl Res.32,529(1963). 3. FRANKLIN, R. M. and BALTIMORE, D., Cold Spring Harbor Symp. Quant. Biol. 27,175 (1962). 4. GAULDEN, M. E. and PERRY, R. P., Proc. Nat/. Acad. Sci. U.S. 44, 553 (1958). 5. GIRARD, &I., PENMAN, S. and DARNELL, J. E., Proc. NatI Acad. Sci. U.S. 51, 205 (1964). 6. GOLDBERG, I. H. and REICH, E., Fed. Proc. 23, 958 (1964). Experimental
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7. HOXIG, G. R. and RABINOVITZ, M., Science 149, 1504 (1965). 8. KILLANDEH, D. and ZETTERBERG, A., E.zpfZ Cell Res. 38, 272 (1965). 9. -Exptl Cell Res. 40, 12 (1965). 10. KISHIMOTO, S. and LIEBERMAN, I., Exptl Cell Res. 36, 92 (1964). 11. KUNTZ, M., J. Gen. Physiol. 33, 349 (1950). 12. LASZLO, J., MILLER, D. S., MCCARTHY, K. S. and HOCHSTEIN, P., Science 151, 1007 (1966). 13. LERMAN, M. I. and BEXYU~~OVICH, M. S., Nature 206, 1231 (1965). 11. LEVINTHAL, C., KEYNAN, A. and HIGA, A., Proc. A’atl. Acad. Sci. U.S. 48, 1631 (1962). 15. MCDONALD, M. R., J. Gen. Physiol. 32, 39 (1948). 16. MAZIA, D., in J. BR~CHET and A. E. MIRSKV (cds.), The Cell, vol. 3, p. 77. Academic Press, New York, 1961. 17. MITA, T., Biochim. Hiophys. Ada 103, 182 (1965). 18. MITTERIAYER, C.. BRAUN. R. and RUSCH. H. P., Expfl Cell Res. 38, 33 (1965). 19. PERRY, R. P.,‘Ex&Z Cell Res. 29, 400 (1963). ’ 20. SCHIFF, S. O., Exptl Cell Res. 40, 264 (1965). 21. SISKEX, J. E., in’D. PRESCOTT (ed.), ifethbds in Cell Physiology, vol. 1, p. 387. Academic Press, New York, 1964. 22. SISKEN, J. E. and KINOSITA, R., .I. Biophys. Biochem. Cytol. 9, 509 (1961). 23. SISKEN, J. E. and MORASC.~, L., J. Cell Biol. 25, 179 (1965). 21. TAYLOR, E. IV., J. Cell Biol. 19, 1 (1963). 25. --Exptl Cell Res. 40, 316 (1965). 26. TOBEY, R. A., PETERSON, D. F., ANDERSON, E. C. and PUCK, T. T.,Biophys. .J. 6, 567(1966). 27. WIIITSON, G. L. and PADILLA, G. M., Exptl Cell Res. 36, 667 (1964). 28. YARMOLINSKY, M. B. and DE LA HABA, G. L.. Proc. Nat1 Acad. Sci. U.S. 45, 1721 (1959). 29. ZETTISRRERG, A. and KILLANDER, D., Exptl Cell Res. 40, 1 (1965).
Experimental
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Press Inc.
Cell Research
46,
93-105
93
(1967)
RNA AND PROTEIN SYNTHESIS REQUIRED FOR ENTRY OF CELLS INTO MITOSIS AND DURING THE MITOTIC CYCLE GRACE Department
of Biology,
M.
DONNELLY City
of Hope
Received
and Medical August
J.
E.
Center,
SISKEN Duarte,
CaliJ,
U.S.A.
29,1966
THE fact that there is a delay between the completion of DNA synthesis and the entry of cells into recognizable mitosis in nearly all somatic cells thus far studied suggests, a priori, that this is a period during which specific metabolic or organizational events related to the ensuing division are occurring. It also suggests that there is some relationship between the completion of DNA synthesis and the initiation of those events which convert an interphase cell into one organized for division. Inhibitors whose primary modes of action in cells are relatively well defined, when used in conjunction with various kinetic techniques, afford a means of studying biochemical events at different times during the mitotic cycle. Thus, by using puromycin, an inhibitor of protein synthesis [as], and actinomycin D (AMD), an inhibitor of DNA-dependent RNA synthesis [S], it is possible to study the requirements for division of the synthesis of nucleic acids and proteins during the predivision period. Indeed, numerous such studies have recently been reported [2, 10, 17, 18, 24-271. However, thus far, none has exploited Perry’s [19] finding that treatment with AMD can lead to qualitatively different effects depending on the levels of the agent utilized; at 4 x 1O-8 M the main inhibitory effect of actinomycin D is on ribosomal RNA synthesized at the nucleolus, while at 4 x 10-s M all RNA synthesis is inhibited. In this study we attempted to gain some insight into the roles of the RNAs synthesized during the predivision period by comparing the effects of these concentrations of actinomycin D to the effects of puromycin and by studying the characteristics of cell populations which had been exposed to actinomycin D for short periods and then returned to normal medium. Contribution No. G2-66, Department of Biology. Supported from the National Cancer Institute, U.S. Public Health Service Grant FR 05471, U.S. Public Health Service.
in part by Grant CA-04526-07 and General Research Support
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Grace M. Donnelly
and J. E. Sisken
METHODS The Fernandes line of human amnion cells was used in all of our experiments. These cells were grown in Rose chambers or on coverslips in Leighton tubes. All coverslips were fixed with two changes of ethanol acetic acid, 3:1, and rinsed with two changes of ethanol. For mitotic index studies, the cells were stained with acetic orcein, dehydrated in ethanol, and mounted with Permount. Coverslips for autoradiographs and liquid scintillation counting were further extracted with several changes of cold 5 per cent TCA followed by several rinses in distilled water. Kodak NTB-2 emulsion was used for autoradiographs which were stained through the film with Azure A after development. Actinomycin D (generously supplied by Dr. George E. Boxer, Merck and Co., Rahway, New Jersey) was used in two concentrations, 4 pg/ml (approximately 4 x 1O-s M) and 0.04 pg/ml (approximately 4 x 1O-8 M). Puromycin was used at a concentration of 25 lug/ml (4.6 x 1O-6 M). Tritiated uridine was used at a concentration of 2 PC/ml (3c/mM). DNAse (Worthington, 1 x crystallized, 0.1 mg/ml, pH 7.5) digestions were performed at room temperature for 3 hr [ll]. RNAse (Worthington, crystallized from EtOH, 2 mg/ml, pH 6) digestions were performed at 45°C for 3 hr [15]. The cinemicrographic method was as previously described [al], using low-power phase objectives and with exposures being made at a rate of one frame per minute. For measurements of gross rates of synthesis, coverslips with labelled cells were crushed in glass counting vials. The cells were solubilized by shaking with Hyamine 10X for l& hr at 37°C. Counting fluid was added and the radioactivity was measured in a liquid scintillation counter. RESULTS The effects of the inhibition
of protein
synthesis
The effects of 4.6x 1W6 M puromycin on three cell parameters are shown in Fig. 1. Curve A shows that this concentration reduces the rate of protein synthesis in our material to about a third of the control rate within 15-30 min, levels off quickly and remains at the 20-25 per cent level for at least 2 hr. No samples were taken beyond this time, since time-lapse motion pictures of cells in Rose chambers showed that cells began to die about 2 hr after the beginning of treatment. The time-lapse films also showed that cells were inhibited from entering mitosis very soon after exposure to this concentration of puromycin. The kinetics of this inhibition are shown by the data of curve B, Fig. 1, which demonstrates that the metaphase index drops very rapidly to about half the control level in 30 min and to essentially zero in 2 hr. Because of the rapidity of this effect and because cells begin to die within 2 hr, the question arises as to whether the agent caused an overall inhibition of cellular metabolic processes, perhaps through an inhibition of some Experimental
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RNA and protein
9,5
synthesis and mitosis
sensitive step in a generally required system (e.g., some rapidly-turning-over respiratory enzyme), or whether the effect has some degree of specificity on the processes required for the entry of cells into division.We therefore studied the effects of puromycin on the gross rate of nucleic acid synthesis and found (Curve C, Fig. 1) that this level of puromycin inhibits the rate of incorporation of 3H-uridine to only a small extent over a 2-hr period. % CONTROL 130
1
c
Fig.
1.
Fig.
2.
Fig. l.-Effects of 4 x 1O-6 M puromycin on human amnion cells. Curve A, Relative rate of incorporation of isotope into replicate coverslip cultures exposed to W-leucine for 15 min prior to fixation at various intervals after the beginning of treatment. Curve B, The mitotic index at various times since exposure of coverslip cultures to puromycin. Each point is based on counts of 9000 cells from at least 2 experiments. The control value is based on counts of 155,000 cells obtained from several different experiments. Curve C, Same as in curve A, except cells were exposed to SH-uridine. In curves A and C, nearly all points are derived from 3 or 4 replicate cultures in each of at least 2 separate experiments. Curve D, A reproduction of curve B, Fig. 2, metaphase index following treatment with 4 pg/ml AMD. Fig. 2.-Actinomycin inhibition of RNA synthesis and mitosis during treatment. The metaphase index was determined for cells grown on coverslips and exposed to 4 fig/ml (Curve B) and 0.04 pg/ml (Curve D) of AMD for varying time periods before fixation. 5000 to 11,000 cells were counted for each time period. The number of metaphases for each 1000 cells was recorded using a double blind scoring method. Controls were averaged and treatments were compared with the overall control mean (17.1kO.64 metaphases per thousand cells) based on 155,000 cells. Other coverslips were similarly exposed to AMD (Curve A, 4 pg/ml; Curve C 0.04 ,og/ml) but then labelled with tritiated uridine (2 PC/ml) in the appropriate concentration of AMD for 15 min prior to fixation. Fixed cells were further extracted with cold 5 per cent TCA and the coverslips were prepared for liquid scintillation counting. Counts/min/sample from tritiated uridine in actinomycin treated samples were compared with counts/min in untreated control samples. Experimental
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Grace M. Donnelly
The effects of the inhibition
and J. l?. Sisken
of RNA synthesis
The effect of the concentrations of AMD used by Perry [19] on the overall rates of incorporation of 3H-uridine into total nucleic acids are shown in Fig. 2, Curves A and C. After only 10 min of treatment with 4 pg/ml AMD, 3H-uridine incorporation had dropped to almost 30 per cent of the control value and was down to 15 per cent of control after 1 hr. With 0.04 pg/ml, the decrease was not nearly so drastic. Samples treated for 10 min had 65 per cent of the control value, and at the end of 3 hr, incorporation was still at 30 per cent of the control level. Control samples which had been RNAase digested following exposure to 3H-uridine in two experiments indicated that 3H-uridine in the DNA fraction contributed 7-10 per cent to the total counts. The differential effects of these concentrations of AMD on nucleolar and non-nucleolar RNA synthesis are indicated by the autoradiographs of Fig 3. Untreated controls showed the expected short term 3H-uridine labelling pattern (Fig. 3A, B, C). Nuclei were well labelled and there was a large population of cells with heavily labelled nucleoli. Grains in the cytoplasm were not above background, and RNAase digestion resulted in autoradiographs with essentially no label above background in about two-thirds of the cells (Fig. 3B). The remaining cells retained some label, presumably in DNA. Cells pretreated with 4 ,ug/ml AMD have all autoradiographically detectable RNA synthesis blocked within 15 min (Fig. 3F). A loo-fold dilution, on the other hand, blocks nucleolar RNA synthesis selectively and quickly, while chromatin RNA synthesis is relatively unaffected (Fig. 3D). The effect of actinomycin on the entry of cells into mitosis is shown by the metaphase index data of Fig. 2, Curves B and 1). With 4 pg/ml AMD (Curve B), there was a surprisingly rapid decline in the metaphase index, one which was almost as rapid as that produced by puromycin (Fig. 1, Curve B). In fact, the difference between the two curves at the 50 per cent level is of the order of only 10 min (Fig. 1, Curve D). The lower concentration of actinomycin produced a different pattern of mitotic inhibition (Fig. 2, Curve D). The metaphase index of populations treated with 0.04 ,ug/ml remained at about the control value for almost 3
Fig. 3.-Autoradiographs of human amnion cells exposed to tritiated uridine following actinomycin treatment. Cells were treated with AMD (4 pg/ml or 0.04 pg/ml) for 15 min and then labelled for 15 min with tritiated uridine (2 PC/ml) in the appropriate concentration of AMD prior to fixation. Experimental
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46
KXA and protein
A, W and B, RKAase ,ug/ml A&ID /lg/ml AMD treated with
C, Untreated controls, showing digested. C, DN\‘Aase digested showing selective loss of label and DNAase digested, showing 4 /cg/ml AMD showing almost
synthesis und miiosis
the typical uridine labelling pattern. A, undigested; showing labelled nucleoli. II, Cells treated with 0.04 associated with nucleoli. I:‘, Cells treated with 0.0-1 presence of non-DIV.4 label in nucleus. I:. Cells total loss of label in both nucleolus and chromatin.
98
Grace M. Donnelly
and J. E. Sisken
hr, and then dropped to 75 per cent of control at the fourth hour. After seven hours of treatment, the metaphase index had fallen to about 20 per cent, and by the tenth hour, the slides were unscorable. There is some variability in the data and it is formally possible to also fit the line E to these same data. The explanation for such a line would be that an initial increase in metaphase index, due to prolongation of metaphase, occurred immediately after the beginning of treatment, but that there was also an immediate inhibitory effect on the entry of cells into metaphase. However, direct observations with time-lapse cinemicrography showed that the continuous treatment of cells with this concentration of AMD over a much longer period has no effect on the duration of metaphase. During a 24 hr control period prior to treatment, metaphase times from three separate experiments averaged 16.4kO.4 min (n=145) while the mean duration of metaphase for periods of up to 7.5 hr of continuous treatment was 16.4+ 1 .O min (n=45). Since metaphase times are not increased, we believe line D presents a more correct interpretation of the effects of 0.04 pg/ml AMD on metaphase index. Time-lapse cinemicrographs showed that actinomycin killed Fernandes human amnion cells quickly. With continuous exposure to 4 ,ug/ml AhID, cells in the colonies under observation began to die within 2-4 hr after the beginning of treatment. By lo-15 hr, the colonies were usually totally obliterated. Very few divisions were seen after the beginning of treatment at this concentration. Those cells that did divide did so within the first 5 hr of treatment and exhibited a prolonged metaphase (Fig. 4). Death, as defined by active “boiling” of the cell and its subsequent “explosion” or collapse, occurred during interphase. With continuous treatment at the lower concentration, deaths began approximately 3 hr after introduction of the drug, and were widespread by the 7th hr. Divisions after treatment were more common than in the colonies treated with the higher concentration, and were noted as late as 20 hr after the beginning of treatment. At approximately 22 hr, almost all cells were dead. Recovery of cells after removal of actinomycin The fact that under continuous treatment metaphase times were not increased and the metaphase index was relatively unaffected by 0.04 ,ug/ml AMD for up to 3 hr (Fig. 2, Curve D), shows that cells in the last 3 hr of interphase (i.e., late S or G2) were not delayed in their entry into mitosis. The studies in this section were designed to determine the effects of a 3 hr Experimental
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RNA and protein
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99
treatment at this lower concentration on other aspects of cellular kinetics. (No further studies were made of the higher concentration because the time-lapse films had shown that removal of actinomycin after 3 hr treatment at 4 ,ug/ml AMD did not result in any recovery, and that cells continued to die, although perhaps at a slightly slower rate.) In the first experiments, populations of cells growing in Rose chambers were followed by time-lapse cinemicrography under control conditions for periods of up to 24 hr in order to record mitotic histories of individual cells. Photography continued during the 3 hr treatment with 0.04 pg/ml AMD as well as during the post-treatment period. Cells were followed for periods of up to 95 hr beyond treatment. The data in Fig. 5 indicate the duration of mitotic cycles of cells born at different times in relation to the period of treatment. Cells 17-20 hr old at the time of treatment, and thus in late S or G2, had a normal cycle time. Progeny of these cells, however, had elongated cycle times. Cells in all other parts of the cycle at the time of treatment had elongated cycle times, as did their progeny. Analysis of the films also revealed that a burst of mitosis occurred 20 hr
Fig. Fig. 4.-Prolongation (arrow) represent class,
4.
7 and
Fig. of metaphase during 5 cells, respectively.
treatment with 4 pg/ml There were no divisions
5.
AMD. Points after treatment beyond the 5th hr treatment
Fig. B.-Effect of a short treatment of actinomycin D on the duration of the mitotic cycle of cells born at different times in relation to the time of treatment. Cells in a randomly dividing population were followed cinemicrographically before, during, and after 3 hr treatment with 0.04 ,ug/ml AMD. The chart shows that the generation times of cells is related to the age of the cell at the time of treatment. The length of the generation time is shown in hours. The number of cells in each age class is given in parentheses. The left end of the bar indicates the time of birth, and the right end the time of division relative to the time of treatment. The standard errors for the length of the generation times is indicated by the line at the right end of the bar. Experimental
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Grace M. Donnelly
100
and J. E. Sisken
after treatment and that there was an accompanying increase in the duration of individual metaphases (Fig. 6, Curve A). .4 subsequent mitotic index study, utilizing coverslip cultures and the techniques as described in Fig. 2, substantiated the existence of the 20.hr post-treatment peak. These data are compared in Fig. 6. Studies of recovery of cells following a 3 hr treatment with the low concentration of AMD revealed that the gross rate of RNA synthesis returned to about 75 per cent of the control level (Fig. 6C) almost immediately after
0
.i b” L0
---L,” 10
20 TIME
jo
-407
IHOURS)
Fig. 6.-Mitotic index, RNA synlhrsis, and duration of metaphase following a short-term treatment of actinomycin D at 0.04 pg/ml. -4, Duration of Metaphase (inner scale, in min); B, mitotic index; C, RNA synthesis.
removal of AMD, but that R?;A synthesis did not return to normal until after the tenth hour. Further increase in rate may simply reflect increase in cell number. This level of recovery is greater than that reported by Taylor [X53, but this is probably a result of the lower concentration of AMD used in our experiment. DISCUSSION
The data presented on the efyects of puromycin on the entry of cells into division are in general agreement with previous studies [lo, 25, 261 which indicate that mammalian cells in culture must synthesize at least some protein to within 30-60 min of metaphase in order to carry out those processes which convert them from interphase to mitotic cells. The fact that nucleic acid synthesis is not greatly affected during the first hour or two of treatment with puromycin suggests that the nucleic acid synthesizing systems as well as the systems supplying energy, precursors, and enzymes for nucleic acid Experimental
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RNA and protein
101
synthesis and mitosis
synthesis, are not nearly so sensitive to the agent as is the entry of cells into division. It further suggests that the entry of cells into division is dependent upon the synthesis of new protein molecules just prior to mitosis. Reports in the literature concerning the last synthesis of RNA required for cells to reach mitosis in cultured mammalian cells are more varied and, in some cases at least, are almost certainly a result of differences in concentration of AMD used by the different investigators. These experiments are summarized in Table I, and may be divided into two groups: those in which the concentration of ARID ranged between 0.025 pg/ml and 0.2 ,ug/ml, which WC will call a low concentration; and those in which the concentration utilized was 2 to 5 pg/ml, which we will call a high concentration. Perry [lg] showed that concentrations of the order of 0.04 pg/ml inhibit the synthesis of mainly ribosomal RNA, whereas 4 pg/ml inhibits all RNA synthesis in HeLa cells. Since our autoradiographs tend to confirm these results for amnion cells, it appears that one may interpret all the data on lovv concentration, except Taylor’s [25], as showing the elrects of the inhibition of ribosomal RNA. The data from high concentration experiments indicate the requirement for some other RNA, perhaps messenger RNA. In all experiments but Taylor’s [a;], where lovv concentrations of AMD were used, RNA synthesis, when measured, was incompletely inhibited. The time beyond which this concentration was no longer efTective in inhibiting the entry of cells into mitosis vvas placed at 2 to 4 hr prior to division. In some cases this coincided with the end of the S period. Taylor’s experiments were exceptional in that he obtained complete inhibition of RNA synthesis with as 0.2 ,ug/ml of AMD but he did not observe an efrect on the mitotic index until 3 hr after the beginning of treatment. \Vhen the effects of low concentrations of AMD are compared with those of puromycin, it is always found (Table I) that the latter affects metaphase index much more rapidly, This observation suggests that the low concentration of actinomycin causes the level of some RNA fraction to decline to a point where the metabolic requirements for entry of cells into division can no longer be met. While there might be alternative explanations, this eflect may occur because of the direct role of the RNA in the synthesis of a required protein. If this be true and, if the only significant effect of the low concentration of AMD is on ribosome synthesis, then it is possible to conclude from our data as well as from the data of others that, in the latter part of interphase a cell has approximately a l-3 hr supply of ribosomes which can be used for the synthesis of proteins required for division. This possibility is consistent with the findings of Gaulden and Perry [4] and Schiff [20] Experimental
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Grace M. Donnelly
102
and J. E. S&ken
that nucleolar function immediately prior to division is not requisite for that mitosis. Further support for this point is presented below. The experiments on the effects of continuous treatment with the low concentration of actinomycin D on the mitotic index show that only cells in the last part of interphase (late S-G2) at the time of treatment can proceed to division, and that they do so at normal rates. When, after 3 hr of treatment, we rinsed the cells and reincubated them in fresh medium, all observable TABLE I. Time between
Duration of G2
Cell type
treatment with actinomycin cells into mitosis. Concentration actinomycin ielm
Reference
LOW
KB
3 hr
Rabbit
kidney
Mouse Mouse Human
fibroblast fibroblast amnion
Min
of 4 hr
0.2
[lo]
0.33
PI PI
0.075 0.025-0.05 0.04
This
3.2 hr
P61
2.2 hr
[II This
paper
HIGH
Chinese Chinese Human
hamster hamster amnion
Degree of inhibition of RNA synthesis
Time between treatment and inhibition of entry into mitosis
CONCENTRATION
WI
2.2 hr
of D
D and efect on entry of
Essentially total in 15 min 90 yh over a 4-hr period
Approx. 80 y0 in 1 hr
3 hr Approx.
4 hr
2 hr “Considerable 3-4 hr
time”
CONCENTRATIOS
paper
2.0 5.0 4.0
85 % m 1 hr Approx. 95 % in 1 hr
1.3 hr Approx. 45 min 30-40 min
cells continued on to division and, in many cases their progeny could be followed into subsequent divisions. Cells which were in early interphase during the treatment required more than the normal amount of time to reach mitosis. Cells which were in late interphase entered mitosis at their normally expected times, but the progeny of these cells had prolonged cycle times which were approximately equal to the cycle time of those cells which had been treated in the early part of their cycle. These results are similar to observations which have been made on slime molds [18] and protozoa [17, 271. The fact that treatment in the late part of one mitotic cycle can affect the subsequent division is explainable in at least two ways. The first possibility Experimental
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RNA
and protein
103
synthesis and mitosis
is simply as suggested above, namely that the ribosomes made in G2 are really required by the cell in its next cycle and, if the cell begins its next cycle with a deficiency of ribosomes, its Gl and cycle time will be prolonged. It has been shown by Killander and Zetterberg [S] that variations in Gl (which in our cell line accounts for most of the variations in duration of the mitotic cycle [22, 231) are dependent upon the mass of a cell at the beginning of interphase, and that cell mass and rate of protein synthesis during the mitotic cycle are correlated with RNA content [9, 291. On the other hand, it has been shown that AMD binds very tightly to DNA [6j, and it is not easily removed by simple washing. This, then, suggests the second possibility which is that, even though cells in G2 at the time of treatment can divide, they still may have bound to their DNA some A1fD that continues to inhibit the synthesis of RNA throughout the next cycle. These hypotheses are not mutually exclusive, and, in reality, both hypotheses are probably correct to at least some extent. In all three experiments with the high concentration of AMD (Table I) the effect on the entry of cells into division occurs between 0.5 and 1.3 hr after the beginning of treatment. Only in our experiments and those of Tobcy et al. [26] may the effects of the high concentration of AMD be compared with those of puromycin. In the latter the differences in time between the effects of the two agents is about 45 min while in our experiments it is of the order of 10-15 min. As indicated by the differences in G2 times of these two cell lines, the 30-min discrepancy could easily be a result of inherent differences in the cell lines utilized. In either case, however, we need to be able to account for the gap in time between the effects of the two agents. It is possible to propose at least four explanations for this gap: (1) The cell in G2 must continually make some messenger RNA which has a very short half-life. From our data, for example, it would be estimated to be something like 10-15 min. While messengers with half-lives of about a minute have been reported for bacterial cells [14], the shortest half-life reported for a mammalian messenger is about 1 hr [5]. (2) S ome division-related messenger is synthesized up to but not beyond a specific time prior to division (45 min in Chinese hamster cells, and lo-15 min in human amnion). It need not have an exceptionally short half-life. This is the kind of hypothesis favoured by Tobey et al. [26]. (3) There is another kind of RN,4 involved which has the characteristic of messenger RNA in being inhibited only by high concentrations of AMD, but it is not a messenger in the usual sense. A possible example of this might be the RNA which is usually found associated with the mitotic apparatus (see review by Mazia, [16]). (4) The rapid effect of the high Experimental
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104
Grace M. Donnelly
and J. E. S&ken
concentration in our system is a result of some side reaction of AMD unrelated to RNA synthesis. Actinomycin at high concentrations has been shown to affect other systems, such as energy metabolism [7, 121 and protein synthesis [3, 13, 251 but, even at very high concentrations, the levels of inhibition observed within the first hour of treatment by these authors do not seem nearly great enough to account for the rapid decrease in mitotic index observed in our experiments. To our knowledge, no critical experiments have been reported which would rule out any of these possibilities.
SUMMARY
As a means of studying metabolic requisites for mitosis, actinomycin D, in two concentrations, and puromycin were compared in their respective inhibitions of RNA and protein synthesis during the predivision period. Treatment of the cells with levels of puromycin which reduce the gross rate of protein synthesis to about 20 per cent of the control level rapidly inhibits the entry of cells into mitosis. In agreement with the findings of others, the kinetics of this effect suggest that new protein molecules must be synthesized within a 30-60 min period prior to metaphase for division to occur. Low concentrations of actinomycin (0.04 pg/ml) which selectively inhibit nucleolar RNA synthesis do not affect the entry of cells into division for periods of up to 3 hr. This suggests that cells in the latter part of the mitotic cycle already contain a l-3 hr supply of ribosomes which can be utilized for the syntheses involved in preparing cells for division. Removal of these low concentrations of AMD from the medium after a 3-hr period allows division to occur, but c,ells treated early in their mitotir cycle require more time to get to division. Cells treated during G2 or late S are not delayed in entering division but their next mitotic cycle is prolonged. A high concentration of AMD (4 pg/ml) affects the entry of cells into division almost as rapidly as puromycin. Several alternative possibilities are presented to account for this finding. REFERENCES 1. ARRIGHI, F. E. and Hsu, T. C., Exptl Cell Res. 39, 305 (1965). 2. CASPERSSON, T., FARBER, S.,FOLEY,G.E. and KILLANDER, D., ExptlCeZl Res.32,529(1963). 3. FRANKLIN, R. M. and BALTIMORE, D., Cold Spring Harbor Symp. Quant. Biol. 27,175 (1962). 4. GAULDEN, M. E. and PERRY, R. P., Proc. Nat/. Acad. Sci. U.S. 44, 553 (1958). 5. GIRARD, &I., PENMAN, S. and DARNELL, J. E., Proc. NatI Acad. Sci. U.S. 51, 205 (1964). 6. GOLDBERG, I. H. and REICH, E., Fed. Proc. 23, 958 (1964). Experimental
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7. HOXIG, G. R. and RABINOVITZ, M., Science 149, 1504 (1965). 8. KILLANDEH, D. and ZETTERBERG, A., E.zpfZ Cell Res. 38, 272 (1965). 9. -Exptl Cell Res. 40, 12 (1965). 10. KISHIMOTO, S. and LIEBERMAN, I., Exptl Cell Res. 36, 92 (1964). 11. KUNTZ, M., J. Gen. Physiol. 33, 349 (1950). 12. LASZLO, J., MILLER, D. S., MCCARTHY, K. S. and HOCHSTEIN, P., Science 151, 1007 (1966). 13. LERMAN, M. I. and BEXYU~~OVICH, M. S., Nature 206, 1231 (1965). 11. LEVINTHAL, C., KEYNAN, A. and HIGA, A., Proc. A’atl. Acad. Sci. U.S. 48, 1631 (1962). 15. MCDONALD, M. R., J. Gen. Physiol. 32, 39 (1948). 16. MAZIA, D., in J. BR~CHET and A. E. MIRSKV (cds.), The Cell, vol. 3, p. 77. Academic Press, New York, 1961. 17. MITA, T., Biochim. Hiophys. Ada 103, 182 (1965). 18. MITTERIAYER, C.. BRAUN. R. and RUSCH. H. P., Expfl Cell Res. 38, 33 (1965). 19. PERRY, R. P.,‘Ex&Z Cell Res. 29, 400 (1963). ’ 20. SCHIFF, S. O., Exptl Cell Res. 40, 264 (1965). 21. SISKEX, J. E., in’D. PRESCOTT (ed.), ifethbds in Cell Physiology, vol. 1, p. 387. Academic Press, New York, 1964. 22. SISKEN, J. E. and KINOSITA, R., .I. Biophys. Biochem. Cytol. 9, 509 (1961). 23. SISKEN, J. E. and MORASC.~, L., J. Cell Biol. 25, 179 (1965). 21. TAYLOR, E. IV., J. Cell Biol. 19, 1 (1963). 25. --Exptl Cell Res. 40, 316 (1965). 26. TOBEY, R. A., PETERSON, D. F., ANDERSON, E. C. and PUCK, T. T.,Biophys. .J. 6, 567(1966). 27. WIIITSON, G. L. and PADILLA, G. M., Exptl Cell Res. 36, 667 (1964). 28. YARMOLINSKY, M. B. and DE LA HABA, G. L.. Proc. Nat1 Acad. Sci. U.S. 45, 1721 (1959). 29. ZETTISRRERG, A. and KILLANDER, D., Exptl Cell Res. 40, 1 (1965).
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